A novel tetraester construct that reduces cationic lipid-associated cytotoxicity. Implications for the onset of cytotoxicity
A novel tetraester construct that reduces cationic lipid-associated cytotoxicity. Implications for the onset of cytotoxicity
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DOI：
10.1021/bi9801154
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发表时间：
1998-05-05
期刊：
影响因子：
2.9
通讯作者：
Nantz, MH
中科院分区：
文献类型：
--
作者：
Aberle, AM;Tablin, F;Nantz, MH
在多核苷酸递送到哺乳动物细胞中期间诱导轻微细胞毒性反应的阳离子两亲物的制备受到酯、酰胺或氨基甲酸酯键的常规使用的限制，所述酯、酰胺或氨基甲酸酯键系链极性或疏水结构域。基于铵的磷酸盐对细胞过程的有害影响已经得到充分证实。本报告是关键四酯构建体的第一个例子，该构建体利用酯键来束缚极性和疏水结构域。以季戊四醇、N，N-二甲基甘氨酸及其相应的脂肪酰基为原料，通过连续的双酯化反应和胺季铵化反应，制备了二肉豆蔻酰和二油酰类似物。使用NIH 3 T3和16 HBE 14（0）-细胞在转染（荧光素酶）和细胞增殖（MTS）测定中检查所得阳离子四酯。四酯在两种细胞系中表现出与充分研究的脂质DOTAP和DC-胆固醇（DC-胆固醇）相当的转染活性。四酯构建体在NIH 3 T3细胞中不提供细胞毒性，并且在16 HBE 14（0）-细胞中提供相对于DC-chol的显著降低的细胞毒性。还使用脂质组检查了两种细胞系中绿色荧光蛋白（GFP）的表达。荧光和相应的相差图像的比较证实了化学细胞毒性结果，并揭示了细胞毒性反应不依赖于转基因表达。在不存在GFP质粒的情况下用阳离子脂质组处理的细胞的相差显微照片显示与GFP转染的细胞相同的形态，表明脂质介导的细胞毒性反应的开始可能发生在内体包封之前的阶段。
The preparation of cationic amphiphiles that induce minor cytotoxic response during polynucleotide delivery into mammalian cells has been limited by the conventional use of ester, amide, or carbamate linkages to tether either the polar or the hydrophobic domains. The deleterious effects of ammonium-based lipidic salts on cellular processes have been well-established. The present report is the first example of a linchpin tetraester construct that utilizes ester linkages to tether both the polar and hydrophobic domains. Dimyristoyl and dioleoyl analogues were prepared from pentaerythritol, N,N-dimethylglycine, and their corresponding fatty acyl groups via successive diesterifications followed by amine quaternization. The resultant cationic tetraesters were examined in transfection (luciferase) and cell proliferation (MTS) assays using NIH 3T3 and 16HBE14(0)-cells. The tetraesters exhibited transfection activity comparable to the well-studied lipids DOTAP and DC-cholesterol (DC-chol) in both cell lines. The tetraester construct afforded no cytotoxicity in NIH3T3 cells and provided a significant lowering of cytotoxicity relative to DC-chol in the 16HBE14(0)-cells. The expression of green fluorescent protein (GFP) in both cell lines also was examined using the lipid panel. Comparison of fluorescent and corresponding phase-contrast images confirmed the chemical cytotoxicity results and revealed that the cytotoxic response was not dependent on transgene expression. Phase-contrast micrographs of cells treated with the cationic lipid panel in the absence of GFP plasmid showed identical morphology to the GFP-transfected cells, suggesting that the onset of a lipid-mediated cytotoxic response might occur at a stage prior to endosomal encapsulation.